This proposal requests a spectral confocal, the Zeiss LSM 510 with Meta detector, to advance our understanding of intracellular infection and host response through the use of novel intracellular reporters and their application to infectious agents present on the NIH list of Priority Pathogens. This instrument has key capabilities that allow quantitative fluorescence of multiple fluors and advanced analysis of data from live samples contained in an environmental chamber. The instrument will be housed in a dedicated Biosafety Level 2 Imaging Center designed specifically for this purpose. We have a group of 10 federally-supported investigators who propose to use these methods to address questions fundamental to intracellular infections. Relevance. The threat from emergent pathogens, and the potential weaponization of infectious agents are issues of which we are increasingly aware. While there have been enormous strides in genomic sequencing of many pathogens, the links between gene and function, and their role in infection are still being determined. Most intracellular pathogens enter their host cells in a membrane-bound compartment of host origin, ie. the phagosome. Under normal circumstances this phagosome would deliver its contents to a lysosome and the pathogen would be killed. But intracellular pathogens avoid this by escaping into the cytosol, remodeling the phagosome, or arresting its maturation. This proposal employs novel fluorescence assays and emerging quantitative fluorescence microscopy to dissect out the infection process for a range of important pathogens. These data will extend ongoing, NIH-funded programs for identification of new drug targets, vaccines and adjuvants. [unreadable] [unreadable] [unreadable]